Staphylococcus aureus is a human pathogen known to infect virtually every organ and cause numerous diseases. Antibiotic-resistant S. aureus strains are rapidly emerging; therefore, new drug targets are being sought. One of the mechanisms utilized by the host to restrict the growth of invading pathogens is the sequestration of the essential nutrient iron within the tetrapyrrole molecule heme. S. aureus employs sophisticated heme uptake systems in order to gain access to this iron source; however, the presence of excess heme is highly toxic to this and many other organisms. The nature of heme toxicity is not fully characterized but is thought to be, at least in part, due to oxidative damage induced by the reactive properties of the iron atom within the heme molecule. S. aureus possesses a heme detoxification system, a predicted transporter named HrtAB, although little is known about its mechanism of action. In the preliminary data presented in this application, we performed a transposon screen in the background of the heme-susceptible hrtA mutant, screening for strains with increased heme-resistance. All mutants identified targeted the menaquinone (MK) biosynthesis pathway. These data suggest that a component of the MK biosynthesis pathway potentiates heme stress. Interestingly, S. aureus strains deficient in MK biosynthesis, known as small colony variants (SCV), are commonly isolated from patients experiencing persistent infections. While multiple factors may contribute to the selection for MK-deficient S. aureus strains, it seems likely that resistance to the toxic effects of heme, an abundant molecule within the vertebrate host, could be playing a role. Based on these data, we predict that conditions of heme stress are directly relevant to the environment experienced by S. aureus during human infection. Therefore, we propose that a thorough understanding of the sources of heme stress in S. aureus and the mechanisms utilized by this pathogen to overcome the toxic effects of heme is essential to advancing our knowledge of staphylococcal physiology within the host environment. To address this issue we propose the following Aims: Aim 1. Determine the mechanism by which MK biosynthesis potentiates heme stress in S. aureus. Aim 2. Define the mechanism of the HrtAB system utilized by S. aureus to alleviate heme toxicity. Previous studies have suggested the use of the MK biosynthesis pathway as a potential antimicrobial target; however, evidence presented in this application indicates that disruption of the MK pathway might selectively induce the formation of persistent and heme-resistant infections. Instead, the heme detoxification system of S. aureus might represent a more viable drug target. By defining the regulation and function of the HrtAB heme detoxification system found in S. aureus and other pathogens such as Bacillus anthracis, Corynebacterium diphtheriae, and Listeria monocytogenes, we will provide potential insight into targeted therapeutic design exploiting the heme-susceptibility of these organisms.